Comparison of permitted daily exposure (PDE) values for active pharmaceutical ingredients (APIs) - Evidence of a robust approach.

Permitted Daily Exposure Limits (PDEs) are set for Active Pharmaceutical Ingredients (APIs) to control cross-contamination when manufacturing medicinal products in shared facilities. With the lack of official PDE lists for pharmaceuticals, PDEs have to be set by each company separately. Although general rules and guidelines for the setting of PDEs exist, inter-company variations in the setting of PDEs occur and are considered acceptable within a certain range. To evaluate the robustness of the PDE approach between different pharmaceutical companies, data on PDE setting of five marketed APIs (amlodipine, hydrochlorothiazide, metformin, morphine, and omeprazole) were collected and compared. Findings show that the variability between PDE values is within acceptable ranges (below 10-fold) for all compounds, with the highest difference for morphine due to different Point of Departures (PODs) and Adjustment Factors (AFs). Factors of PDE variability identified and further discussed are: (1) availability of data, (2) selection of POD, (3) assignment of AFs, (4) route-to-route extrapolation, and (5) expert judgement and differences in company policies. We conclude that the investigated PDE methods and calculations are robust and scientifically defensible. Additionally, we provide further recommendations to harmonize PDE calculation approaches across the pharmaceutical industry.

Optimization of the TeraTox Assay for Preclinical Teratogenicity Assessment.

Current animal-free methods to assess teratogenicity of drugs under development still deliver high numbers of false negatives. To improve the sensitivity of human teratogenicity prediction, we characterized the TeraTox test, a newly developed multilineage differentiation assay using 3D human-induced pluripotent stem cells. TeraTox produces primary output concentration-dependent cytotoxicity and altered gene expression induced by each test compound. These data are fed into an interpretable machine-learning model to perform prediction, which relates to the concentration-dependent human teratogenicity potential of drug candidates. We applied TeraTox to profile 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. Comparing TeraTox predictions with known human or animal toxicity, we report an accuracy of 69% (specificity: 53%, sensitivity: 79%). TeraTox performed better than 2 quantitative structure-activity relationship models and had a higher sensitivity than the murine embryonic stem cell test (accuracy: 58%, specificity: 76%, and sensitivity: 46%) run in the same laboratory. The overall prediction accuracy could be further improved by combining TeraTox and mouse embryonic stem cell test results. Furthermore, patterns of altered gene expression revealed by TeraTox may help grouping toxicologically similar compounds and possibly deducing common modes of action. The TeraTox assay and the dataset described here therefore represent a new tool and a valuable resource for drug teratogenicity assessment.

Review of embryo-fetal developmental toxicity studies performed for pharmaceuticals approved by FDA in 2018 and 2019.

Details of embryo-fetal development (EFD) studies were compiled for all FDA drug approvals in 2018-19. EFD studies were performed for 82 % of approvals (84 % of small molecules and 70 % of biopharmaceuticals). Rats and rabbits were used for 84 % of small molecule (SM) drugs for which EFD studies were submitted. There was at least a 2-fold difference in sensitivity between the rat and the rabbit relative to the human exposure for the majority of drugs (62 %, small molecules and biopharmaceuticals combined) tested in both species. On average, however, the rat and rabbit were equally sensitive to developmental toxicity. Over the last 2 years, the use of non-human primates (NHP) for the developmental toxicity testing of biopharmaceuticals has fallen (26 % of biologics license applications), with many more biopharmaceuticals now tested in rodents (44 % of BLAs). EFD studies were not required for oncology drugs when the mode of action was associated with known developmental risk. One-third of SM non-oncology drugs and two-thirds of SM oncology drugs induced dysmorphogenesis in at least one species. The newly revised ICH S5(R3) guideline will bring about changes to the design of future EFD studies, particularly with respect to high dose selection. The revised guideline will also influence the interpretation of the findings in EFD studies (e.g. fetal morphological variations) and risk assessment.

Focus on germ-layer markers: A human stem cell-based model for in vitro teratogenicity testing.

Human induced pluripotent stem cells (hiPSC) were used to develop an assay format that may deliver information on teratogenicity of drugs. A human pluripotent stem cell scorecard panel was used to monitor the expression of 96 marker genes that are indicative of the stem cell state or differentiation into the ectoderm, mesoderm and endoderm lineages. We selected a human episomal iPS cell line for the assay based on karyotype stability, initial pluripotency, differentiation capacity and overall gene expression variability. The assay is based on embryoid body formation and was developed to be simply automated. In this proof of concept study, we used eight reference compounds (valproic acid, all-trans-retinoic acid, thalidomide, methotrexate, hydroxyurea, ascorbic acid, penicillin G and ibuprofen) to test the technical performance of the assay (readout stability) in concentration-response and time-course experiments. We also found that each compound affected marker gene expression in a different way. Various forms of data analysis identified 19 out of 96 early developmental genes as potential predictive markers for teratogenicity. Machine-learning models were run to exemplify how the assay will be developed further. The preliminary results from these analyses suggest that the assay could be suitable for the pre-screening of candidate pharmaceutical compounds. The approach presented here points a way towards development of a human cell-based assay that could replace the murine EST currently used to screen for early indications of potential teratogenicity of drug candidates.

StemBANCC: Governing Access to Material and Data in a Large Stem Cell Research Consortium.

This paper makes the case for implementing an internal governance framework for sharing materials and data in stem cell research consortia. A governance framework can facilitate a transparent and accountable system while building trust among partner institutions. However, avoiding excessive bureaucracy is essential. The development and implementation of a governance framework for materials and data access in the Stem cells for Biological Assays of Novel drugs and prediCtive toxiCology (StemBANCC) consortium is presented as a practical example. The StemBANCC project is a multi-partner European research consortium, which aims to build a resource of 1,500 well characterised induced pluripotent stem cell (iPSC) lines for in vitro disease modelling and toxicology studies. The project governance framework was developed in two stages. A small working group identified key components of a framework and translated the project legal agreements into a draft policy document. The second phase allowed input from all consortium partners to shape the iterative development of a final policy document that could be agreed by all parties. Careful time management strategies were needed to manage the duration of this component. This part of the process also served as an exploratory space where different options could be proposed, potential gaps in planning identified, and project co-ordination activities specified.

Metabotropic glutamate receptor 5 negative allosteric modulators: discovery of 2-chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (basimglurant, RO4917523), a promising novel medicine for psychiatric diseases.

Negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGlu5) have potential for the treatment of psychiatric diseases including depression, fragile X syndrome (FXS), anxiety, obsessive-compulsive disorders, and levodopa induced dyskinesia in Parkinson's disease. Herein we report the optimization of a weakly active screening hit 1 to the potent and selective compounds chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (basimglurant, 2) and 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP, 3). Compound 2 is active in a broad range of anxiety tests reaching the same efficacy but at a 10- to 100-fold lower dose compared to diazepam and is characterized by favorable DMPK properties in rat and monkey as well as an excellent preclinical safety profile and is currently in phase II clinical studies for the treatment of depression and fragile X syndrome. Analogue 3 is the first reported mGlu5 NAM with a long half-life in rodents and is therefore an ideal tool compound for chronic studies in mice and rats.

Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests.

A detailed report is presented on the performance of the embryonic stem cell test (EST) in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the EST can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.

The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods.

From 1996 to 2000, ZEBET (Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments at the BgVV, Berlin, Germany) coordinated the European Centre for the Validation of Alternative Methods (ECVAM) prevalidation and validation study on three embryotoxicity tests: a) a test employing embryonic stem cell lines (EST); b) the micromass (MM) test; and c) the postimplantation rat whole-embryo culture assay (WEC test). The main objectives of the study were to assess the performance of these three in vitro tests in discriminating between non- embryotoxic, weakly embryotoxic and strongly embryotoxic compounds. Phase I of the study (1997) was designed as a prevalidation phase, for test protocol optimisation, and for the establishment of a comprehensive database of in vivo and in vitro data on embryotoxic compounds. Phase II (1998-2000) involved a formal validation trial, conducted under blind conditions on 20 test compounds selected from the database, which were coded and distributed to the participating laboratories. In the preliminary phase of the validation study, six chemicals out of the 20, which showed embryotoxic potential, were tested. These results were used to define new biostatistically based prediction models (PMs) for the MM and WEC tests, and to evaluate those developed previously for the EST. As a next step, the PMs were evaluated by using the results for the remaining 14 chemicals of the definitive phase of the validation study. The three in vitro embryotoxicity tests proved to be applicable to testing a diverse group of chemicals with different embryotoxic potentials (non-embryotoxic, weakly embryotoxic, and strongly embryotoxic). The reproducibility of the three in vitro embryotoxicity tests were acceptable according to the acceptance criteria defined by the Management Team. The concordances between the embryotoxic potentials derived from the in vitro data and from the in vivo data were good for the EST and the WEC (PM2) test, and sufficient for the MM test and the WEC (PM1) tests according to the performance criteria defined by the Management Team before the formal validation study. When applying the PM of the EST to the in vitro data obtained in the definitive phase of the formal validation study, chemicals were classified correctly in 78% of the experiments. For the MM and the WEC tests, the PMs provided 70% and 80% (PM2) correct classifications, respectively. And, very importantly, an excellent predictivity (100%, except for PM1 of the WEC test, with 79%, considered as good) was obtained with strong embryotoxic chemicals in each of the three in vitro tests.

Validation of the postimplantation rat whole-embryo culture test in the international ECVAM validation study on three in vitro embryotoxicity tests.

A detailed report is presented on the performance of the postimplantation rat whole-embryo culture (WEC) test in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the WEC test can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.

The embryonic stem cell test for the early selection of pharmaceutical compounds.

Potential teratogenicity is a major consideration in the development of pharmaceutical substances. Currently its assessment involves large numbers of animal tests at high cost. This study assessed the feasibility of using the embryonic stem cell test (EST), validated by ECVAM in 1999, as a tool for the prediction of embryonic toxicity of pharmaceutical substances early in their development programmes. ESTs were carried out on 6 chemicals with well established toxicity characteristics established from literature and from the ECVAM study, and then on 10 Roche internal pharmaceutical substances already tested in vivo. The model correctly classified 81% of the substances. Further experiments are necessary to increase the database of the assay.